The killer phenotype in the eucaryote Saccharomyces cerevisiae depends on the expression of genes residing in three distinct genomes; the nuclear genome and two cytoplasmically inherited dsRNA genomes. The two dsRNA genomes (L and M) are separately encapsulated in proteinaceous isometric particles which have properties intermediate between viruses and plasmids. All killer strains contain both the M and L genomes. Loss of M results in the loss of killer phenotype. The presence of L and the functions of at least 20 nuclear genes are required for the maintenance of M. Two other classes of nuclear gene mutations affect killing or immunity functions but not the maintenance of M. The immediate aim of this proposed research is to determine which of the three genomes, the L-dsRNA, M-dsRNA, or nuclear genome, code for each of the various polypeptides involved in toxin production, immunity to toxin, plasmid particle structure and replication and transcription of the two dsRNA genomes. To accomplish this mapping study I propose four main approaches: a new direct method of in vitro translational decoding of the dsRNA genomes, in vitro translation of in vivo produced mRNAs encoding killer system polypeptides, size fractionation of the in vivo produced mRNAs, and hybridization challenge of the in vivo produced mRNAs to each of the dsRNA genomes. The proposed studies will make use of existing deletion mutants of the M-dsRNA genome in order to ascertain what genetic information is missing. In vitro translation coupled with immunoprecipitation assays will be used to determine whether nuclear genome kex and rex mutants are blocked at, before, or after synthesis of toxin and immunity polypeptides, respectively. I anticipate that these studies will result in a description of the major features of gene expression in the yeast killer system. The information gained will contribute to an understanding of the functional interrelationships between the two cytoplasmic dsRNA genomes and between these genomes and the yeast nuclear DNA genome. This knowledge should extend our concepts of nucleocytoplasmic relationships in eucaryotes in general.